Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences.

Abstract

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5-and 10-fold-lower affinity to ARS) and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purified OBFI to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.The replication of eucaryotic chromosomal DNA proceeds by numerous initiations along a chromosome, generating multiple replicons. It is believed that in Sacc haromyces cerevisiae the initiation sites at replicons are specific and correspond to the autonomously replicating sequences (ARSs). ARSs were isolated as DNA fragments that confer on plasmids bearing them the ability to replicate autonomously in yeast cells (29). Both in vitro (6,13,16) and in vivo (3,11,19,23)

Genome-wide Analysis of ARS (Autonomously Replicating Sequence) Binding Factor 1 (Abf1p)-mediated Transcriptional Regulation in Saccharomyces cerevisiae

“…For instance, the K625I mutation in the context of aa 1-662 results in a major defect in Abf1p nuclear localization and the corresponding mutant protein is grossly overexpressed. 2 In addition, the observation that more Abf1p-(1-643) is associated with endogenous promoters than the wild-type protein (Fig. 3) is also consistent with Abf1p-mediated negative autoregulation.…”

“…Among all of the abf1 mutant alleles examined in the study, a point mutation in CS1-(1-662/ K625I) that resulted in impaired nuclear localization exhibited the most striking phenotypes at both temperatures (lanes 5 and 12). 2 For instance, the Abf1p-repressed genes in the Abf1p-(1-662/K625I) mutant background were expressed at levels even higher than those in abf1-1. On the other hand, Abf1p-activated gene expression was significantly compromised in the 1-662/K625I mutant strain with some of the genes in this group expressed below detectable levels (e.g.…”

“…1 -binding factor-1 (Abf1p) is an abundant, essential DNA-binding protein in Saccharomyces cerevisiae (1)(2)(3)(4). Its DNA-binding sites have been found at numerous locations in the yeast genome including the silent mating type loci, ARSs, telomeric X-regions, and the promoter regions of over 80 genes (5,6) (also reviewed in Ref.…”

ARS binding factor I of the yeast Saccharomyces cerevisiae binds to sequences in telomeric and nontelomeric autonomously replicating sequences.

“…This sequence analysis indicated that, contrary to earlier belief, ABFI and OBF1 both bind to the same consensus sequence that has been defined as the consensus ABFI binding sequence (9). It should be pointed out that the purification of the OBF1 protein was carried out by affinity chromatography on immobilized DNA that essentially contained multiple copies of this consensus ABFI binding sequence (20).…”

“…However, the possibility of the existence of a family of such ABFs could not be eliminated at the present time. As far as the identity between ABFI and OBF1 is concerned, Francesconi and Eisenberg (20) indicated that the purified OBF1 protein also bound to ARSI and HMRE-ARS; thus, the possibility exists that the ABFI and OBF1 proteins are similar. This proposal is strongly supported by the results presented in this report.…”